Circular RNA YAP1 acts as the sponge of microRNA‐21‐5p to secure HK‐2 cells from ischaemia/reperfusion‐induced injury

Circular RNA YAP1 (circYAP1) was reported to participate in progression of gastric cancer. However, the role of circYAP1 in acute kidney injury (AKI) remains obscure. We attempted to examine the effects of circYAP1 on ischaemia/reperfusion‐stimulated renal injury. AKI model was established by treating HK‐2 cells in ischaemia/reperfusion (I/R) environment. CircYAP1 expression in blood of AKI patients and I/R‐treated HK‐2 cells was evaluated via RT‐qPCR. CCK‐8, flow cytometry, ELISA and ROS assay were executed to test the impact of circYAP1 on cell viability, apoptosis, inflammatory cytokines and ROS generation. Bioinformatic analysis was executed to explore miRNA targets. The relativity between circYAP1 and miR‐21‐5p was verified by RT‐qPCR and luciferase assay. The functions of miR‐21‐5p in I/R‐triggered injury were reassessed. PI3K/AKT/mTOR pathway was detected by Western blot. Down‐regulated circYAP1 was observed in AKI blood samples and I/R‐treated HK‐2 cells. CircYAP1 overexpression expedited cell growth and weakened secretion of inflammatory factors and ROS generation in I/R‐disposed cells. Besides, we found circYAP1 could sponge to miR‐21‐5p. Interestingly, miR‐21‐5p overexpression overturned the repressive effects of circYAP1 on cell injury. Moreover, PI3K/AKT/mTOR pathway was activated by circYAP1 via inhibiting miR‐21‐5p. We demonstrated that circYAP1 activated PI3K/AKT/mTOR pathway and secured HK‐2 cells from I/R injury via sponging miR‐21‐5p.     

Suppression of epidermal growth factor receptor‐mediated β‐catenin nuclear accumulation enhances the anti‐tumor activity of phosphoinositide 3‐kinase inhibitor in breast cancer

Phosphoinositide 3‐kinase (PI3K) signaling is frequently deregulated in breast cancer and plays a critical role in tumor progression. However, resistance to PI3K inhibitors in breast cancer has emerged, which is due to the enhanced β‐catenin nuclear accumulation. Until now, the mechanisms underlying PI3K inhibition‐induced β‐catenin nuclear accumulation remains largely unknown. In the present study, we found inhibition of PI3K with LY294002 promoted β‐catenin nuclear accumulation in MCF‐7 and MDA‐MB‐231 breast cancer cells. Combining PI3K inhibitor LY294002 with XAV‐939, an inhibitor against β‐catenin nuclear accumulation, produced an additive anti‐proliferation effect against breast cancer cells. Subsequent experiments suggested β‐catenin nuclear accumulation induced by PI3K inhibition depended on the feedback activation of epidermal growth factor receptor (EGFR) signaling pathway in breast cancer cells. Inhibition of EGFR phosphorylation with Gefitinib enhanced anti‐proliferation effect of PI3K inhibitor LY294002 in MCF‐7 and MDA‐MB‐231 cells. Taken together, our findings may elucidate a possible mechanism explaining the poor outcome of PI3K inhibitors in breast cancer treatment.     

Inhibition of the PI3K/AKT pathway potentiates cytotoxicity of EGFR kinase inhibitors in triple‐negative breast cancer cells

Triple‐negative breast cancers (TNBCs) are known to be intrinsically resistant to inhibitors for epidermal growth factor receptor (EGFR). Until now, clinical trials for TNBCs using EGFR inhibitors (EGFRis) as single agents have yielded disappointing results. Here, we report that combinatorial treatment using EGFRis, such as gefitinib or erlotinib, with PI3K/AKT pathway inhibitors (PI3K/AKTis) demonstrated a synergistic, anti‐proliferative effect in cell lines of the basal‐like (BL) subtype, a subtype of TNBC. Western blot analysis revealed that the gefitinib/PI‐103 combination significantly reduced the level of both phospho‐AKT and phospho‐ERK in two susceptible BL subtype cell lines, SUM149PT and MDA‐MB‐468, whereas it had little or no effect on the level of phospho‐ERK in two non‐susceptible cell lines (HS578T and MDA‐MB‐231) of mesenchymal stem‐like (MSL) TNBC subtype. The gefitinib/PI‐103 combination also significantly induced caspase‐3/7‐mediated PARP cleavage and reduced two anti‐apoptotic proteins, XIAP and Bcl‐2 in the susceptible cell lines. In addition, the level of myeloid cell leukemia 1 (Mcl‐1) protein was markedly decreased by gefitinib/PI‐103 combination in the BL TNBC cells, but showed no significant change by this combination in MSL subtype cells. These results suggest that pharmacological inhibition of EGFR used in combination of PI3K/AKTis is a potential therapeutic approach to treat a subtype of TNBCs.     

Positive feedback in Cav‐1‐ROS signalling in PSCs mediates metabolic coupling between PSCs and tumour cells

Caveolin‐1 (Cav‐1) is the principal structural component of caveolae, and its dysregulation occurs in cancer. However, the role of Cav‐1 in pancreatic cancer (PDAC) tumorigenesis and metabolism is largely unknown. In this study, we aimed to investigate the effect of pancreatic stellate cell (PSC) Cav‐1 on PDAC metabolism and aggression. We found that Cav‐1 is expressed at low levels in PDAC stroma and that the loss of stromal Cav‐1 is associated with poor survival. In PSCs, knockdown of Cav‐1 promoted the production of reactive oxygen species (ROS), while ROS production further reduced the expression of Cav‐1. Positive feedback occurs in Cav‐1‐ROS signalling in PSCs, which promotes PDAC growth and induces stroma‐tumour metabolic coupling in PDAC. In PSCs, positive feedback in Cav‐1‐ROS signalling induced a shift in energy metabolism to glycolysis, with up‐regulated expression of glycolytic enzymes (hexokinase 2 (HK‐2), 6‐phosphofructokinase (PFKP) and pyruvate kinase isozyme type M2 (PKM2)) and transporter (Glut1) expression and down‐regulated expression of oxidative phosphorylation (OXPHOS) enzymes (translocase of outer mitochondrial membrane 20 (TOMM20) and NAD(P)H dehydrogenase [quinone] 1 (NQO1)). These events resulted in high levels of glycolysis products such as lactate, which was secreted by up‐regulated monocarboxylate transporter 4 (MCT4) in PSCs. Simultaneously, PDAC cells took up these glycolysis products (lactate) through up‐regulated MCT1 to undergo OXPHOS, with down‐regulated expression of glycolytic enzymes (HK‐2, PFKP and PKM2) and up‐regulated expression of OXPHOS enzymes (TOMM20 and NQO1). Interrupting the metabolic coupling between the stroma and tumour cells may be an effective method for tumour therapy.     

Corilagin suppresses RANKL‐induced osteoclastogenesis and inhibits oestrogen deficiency‐induced bone loss via the NF‐κB and PI3K/AKT signalling pathways

Over‐activated osteoclastogenesis, which is initiated by inflammation, has been implicated in osteoporosis. Corilagin, a natural compound extracted from various medicinal herbaceous plants, such as Cinnamomum cassia, has antioxidant and anti‐inflammatory activities. We found that Corilagin suppressed osteoclast differentiation in a dose‐dependent manner, significantly decreased osteoclast‐related gene expression and impaired bone resorption by osteoclasts. Moreover, phosphorylation of members of the nuclear factor‐kappaB (NF‐κB) and PI3K/AKT signalling pathways was reduced by Corilagin. In a murine model of osteoporosis, Corilagin inhibited osteoclast functions in vivo and restored oestrogen deficiency‐induced bone loss. In conclusion, our findings suggested that Corilagin inhibited osteoclastogenesis by down‐regulating the NF‐κB and PI3K/AKT signalling pathways, thus showing its potential possibility for the treatment of osteoporosis.     

Aromatic‐turmerone attenuates invasion and expression of MMP‐9 and COX‐2 through inhibition of NF‐κB activation in TPA‐induced breast cancer cells

Recent evidence suggests that breast cancer is one of the most common forms of malignancy in females, and metastasis from the primary cancer site is the main cause of death. Aromatic (ar)‐turmerone is present in Curcuma longa and is a common remedy and food. In the present study, we investigated the inhibitory effects of ar‐turmerone on expression and enzymatic activity levels of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced matrix metalloproteinase (MMP)‐9 and cyclooxygenaase‐2 (COX‐2) in breast cancer cells. Our data indicated that ar‐turmerone treatment significantly inhibited enzymatic activity and expression of MMP‐9 and COX‐2 at non‐cytotoxic concentrations. However, the expression of tissue inhibitor of metalloproteinase (TIMP)‐1, TIMP‐2, MMP‐2, and COX‐1 did not change upon ar‐turmerone treatment. We found that ar‐turmerone inhibited the activation of NF‐κB, whereas it did not affect AP‐1 activation. Moreover, The ChIP assay revealed that in vivo binding activities of NF‐κB to the MMP‐9 and COX‐2 promoter were significantly inhibited by ar‐turmerone. Our data showed that ar‐turmerone reduced the phosphorylation of PI3K/Akt and ERK1/2 signaling, whereas it did not affect phosphorylation of JNK or p38 MAPK. Thus, transfection of breast cancer cells with PI3K/Akt and ERK1/2 siRNAs significantly decreased TPA‐induced MMP‐9 and COX‐2 expression. These results suggest that ar‐turmerone suppressed the TPA‐induced up‐regulation of MMP‐9 and COX‐2 expression by blocking NF‐κB, PI3K/Akt, and ERK1/2 signaling in human breast cancer cells. Furthermore, ar‐turmerone significantly inhibited TPA‐induced invasion, migration, and colony formation in human breast cancer cells. J. Cell. Biochem. 113: 3653–3662, 2012. © 2012 Wiley Periodicals, Inc.     

Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway

Acetyl‐11‐keto‐β‐boswellic acid (AKBA), an active triterpenoid compound from the extract of Boswellia serrate, has been reported previously in our group to alleviate fibrosis in vascular remodelling. This study aimed to elucidate the in vivo and in vitro efficacy and mechanism of AKBA in renal interstitial fibrosis. The experimental renal fibrosis was produced in C57BL/6 mice via unilateral ureteral obstruction (UUO). Hypoxia‐induced HK‐2 cells were used to imitate the pathological process of renal fibrosis in vitro. Results showed that the treatment of AKBA significantly alleviated UUO‐induced impairment of renal function and improved the renal fibrosis by decreasing the expression of TGF‐β1, α‐SMA, collagen I and collagen IV in UUO kidneys. In hypoxia‐induced HK‐2 cells, AKBA displayed remarkable cell protective effects and anti‐fibrotic properties by increasing the cell viability, decreasing the lactate dehydrogenase (LDH) release and inhibiting fibrotic factor expression. Moreover, in obstructed kidneys and HK‐2 cells, AKBA markedly down‐regulated the expression of TGFβ‐RI, TGFβ‐RII, phosphorylated‐Smad2/3 (p‐Smad2/3) and Smad4 in a dose‐dependent fashion while up‐regulated the expression of Klotho and Smad7 in the same manner. In addition, the effects of AKBA on the Klotho/TGF‐β/Smad signalling were reversed by transfecting with siRNA‐Klotho in HK‐2 cells. In conclusion, our findings provide evidence that AKBA can effectively protect kidney against interstitial fibrosis, and this renoprotective effect involves the Klotho/TGF‐β/Smad signalling pathway. Therefore, AKBA could be considered as a promising candidate drug for renal interstitial fibrosis.     

AMPH1 functions as a tumour suppressor in ovarian cancer via the inactivation of PI3K/AKT pathway

AMPH1, an abundant protein in nerve terminals, plays a critical role in the recruitment of dynamin to sites of clathrin‐mediated endocytosis. Recently, it is reported to be involved in breast cancer and lung cancer. However, the impact of AMPH1 on ovarian cancer is unclear. In this study, we used gain‐of‐function and loss‐of‐function methods to explore the role of AMPH1 in ovarian cancer cells. AMPH1 inhibited ovarian cancer cell growth and cell migration, and promoted caspase‐3 activity, resulting in the increase of cell apoptosis. In xenograft mice model, AMPH1 prevented tumour progression. The anti‐oncogene effects of AMPH1 on ovarian cancer might be partially due to the inhibition of PI3K/AKT signalling pathway after overexpression of AMPH1. Immunohistochemistry analysis showed that the staining of AMPH1 was remarkably reduced in ovarian cancer tissues compared with normal ovarian tissues. In conclusion, our study identifies AMPH1 as a tumour suppressor in ovarian cancer in vitro and in vivo. This is the first evidence that AMPH1 inhibited cell growth and migration, and induced apoptosis via the inactivation of PI3K/AKT signalling pathway on ovarian cancer, which may be used as an effective strategy.     

ROS accumulation contributes to abamectin‐induced apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway in TM3 Leydig cells

Abamectin (ABA) as one of the worldwide used compounds in agriculture has raised safety concerns on nontarget organism toxicity. However, the study of male reproductive system damage caused by ABA remains unclear. Our aim is to investigate the effect of ABA‐induced cytotoxicity in TM3 Leydig cells and their underlying mechanisms. ABA inhibits TM3 cell viability and proliferation via cell cycle arrested in the G0/G1 phase. In addition, ABA‐induced mitochondrial depolarization leads to an imbalance in Bcl‐2 family expression, causing caspase‐dependent apoptosis in TM3 cells. The increased ratio of cells expression LC3 protein and LC3‐II to LC3‐I indicated the activation of autophagy potentially. Further experiments revealed ABA treatment reduced phosphatidylinositol 3‐kinase (PI3K), protein kinase B (AKT) phosphorylation, and mammalian target of rapamycin (mTOR) phosphorylation. Pretreatment with a PI3K/AKT inhibitor, LY294002, mimicked the ABA‐mediated effects on cytotoxicity. Pretreatment with a PI3K/AKT agonist, insulin‐like growth factor‐1, reversed the effects of ABA. ABA caused the accumulation of intracellular reactive oxygen species (ROS) by increased intensity of the ROS indicator. However, N‐acetylcysteine as ROS scavengers inhibited ABA‐induced apoptosis and autophagy and reversed these ABA‐mediated effects on PI3K/AKT/mTOR pathway. On the basis of the above results, it is suggested that ABA exposure induces apoptosis and autophagy in TM3 cells by ROS accumulation to mediate PI3K/AKT/mTOR signaling pathway suppression.     

PER1 suppresses glycolysis and cell proliferation in oral squamous cell carcinoma via the PER1/RACK1/PI3K signaling complex

There is increasing evidence that the core clock gene Period 1 (PER1) plays important roles in the formation of various tumors. However, the biological functions and mechanism of PER1 in promoting tumor progression remain largely unknown. Here, we discovered that PER1 was markedly downregulated in oral squamous cell carcinoma (OSCC). Then, OSCC cell lines with stable overexpression, knockdown, and mutation of PER1 were established. We found that PER1 overexpression significantly inhibited glycolysis, glucose uptake, proliferation, and the PI3K/AKT pathway in OSCC cells. The opposite effects were observed in PER1-knockdown OSCC cells. After treatment of PER1-overexpressing OSCC cells with an AKT activator or treatment of PER1-knockdown OSCC cells with an AKT inhibitor, glycolysis, glucose uptake, and proliferation were markedly rescued. In addition, after treatment of PER1-knockdown OSCC cells with a glycolysis inhibitor, the increase in cell proliferation was significantly reversed. Further, coimmunoprecipitation (Co-IP) and cycloheximide (CHX) chase experiment demonstrated that PER1 can bind with RACK1 and PI3K to form the PER1/RACK1/PI3K complex in OSCC cells. In PER1-overexpressing OSCC cells, the abundance of the PER1/RACK1/PI3K complex was significantly increased, the half-life of PI3K was markedly decreased, and glycolysis, proliferation, and the PI3K/AKT pathway were significantly inhibited. However, these effects were markedly reversed in PER1-mutant OSCC cells. In vivo tumorigenicity assays confirmed that PER1 overexpression inhibited tumor growth while suppressing glycolysis, proliferation, and the PI3K/AKT pathway. Collectively, this study generated the novel findings that PER1 suppresses OSCC progression by inhibiting glycolysis-mediated cell proliferation via the formation of the PER1/RACK1/PI3K complex to regulate the stability of PI3K and the PI3K/AKT pathway-dependent manner and that PER1 could potentially be a valuable therapeutic target in OSCC.Subject terms: Oral cancer, Cell growth, RNAi     

Endoplasmic reticulum stress‐induced exosomal miR‐27a‐3p promotes immune escape in breast cancer via regulating PD‐L1 expression in macrophages

Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA‐mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)‐27a‐3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR‐27a‐3p and MAGI2 was predicted using bioinformatic analysis and verified by dual‐luciferase reporter assay. Ectopic expression and inhibition of miR‐27a‐3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co‐cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD‐L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR‐27a‐3p expression. Elevation of miR‐27a‐3p and PD‐L1 levels in macrophages was observed in response to exosomes‐overexpressing miR‐27a‐3p in vivo and in vitro. miR‐27a‐3p could target and negatively regulate MAGI2, while MAGI2 down‐regulated PD‐L1 by up‐regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4⁺, CD8⁺ T cells and IL‐2, and T cells apoptosis were observed in response to co‐culture of macrophages and CD3⁺ T cells. Conjointly, exosomal miR‐27a‐3p promotes immune evasion by up‐regulating PD‐L1 via MAGI2/PTEN/PI3K axis in breast cancer.     

MICAL1 facilitates breast cancer cell proliferation via ROS‐sensitive ERK/cyclin D pathway

Molecule interacting with CasL 1 (MICAL1) is a multidomain flavoprotein mono‐oxygenase that strongly involves in cytoskeleton dynamics and cell oxidoreduction metabolism. Recently, results from our laboratory have shown that MICAL1 modulates reactive oxygen species (ROS) production, and the latter then activates phosphatidyl inositol 3‐kinase (PI3K)/protein kinase B (Akt) signalling pathway which regulates breast cancer cell invasion. Herein, we performed this study to assess the involvement of MICAL1 in breast cancer cell proliferation and to explore the potential molecular mechanism. We noticed that depletion of MICAL1 markedly reduced cell proliferation in breast cancer cell line MCF‐7 and T47D. This effect of MICAL1 on proliferation was independent of wnt/β‐catenin and NF‐κB pathways. Interestingly, depletion of MICAL1 significantly inhibited ROS production, decreased p‐ERK expression and unfavourable for proliferative phenotype of breast cancer cells. Likewise, MICAL1 overexpression increased p‐ERK level as well as p‐ERK nucleus translocation. Moreover, we investigated the effect of MICAL1 on cell cycle‐related proteins. MICAL1 positively regulated CDK4 and cyclin D expression, but not CDK2, CDK6, cyclin A and cyclin E. In addition, more expression of CDK4 and cyclin D by MICAL1 overexpression was blocked by PI3K/Akt inhibitor LY294002. LY294002 treatment also attenuated the increase in the p‐ERK level in MICAL1‐overexpressed breast cancer cells. Together, our results suggest that MICAL1 exhibits its effect on proliferation via maintaining cyclin D expression through ROS‐sensitive PI3K/Akt/ERK signalling in breast cancer cells.     

Inhibition of Orai1‐mediated Ca2+ entry enhances chemosensitivity of HepG2 hepatocarcinoma cells to 5‐fluorouracil

Increasing evidence supports that activation of store‐operated Ca²⁺ entry (SOCE) is implicated in the chemoresistance of cancer cells subjected to chemotherapy. However, the molecular mechanisms underlying chemoresistance are not well understood. In this study, we aim to investigate whether 5‐FU induces hepatocarcinoma cell death through regulating Ca²⁺‐dependent autophagy. [Ca²⁺]_i was measured using fura2/AM dye. Protein expression was determined by Western blotting and immunohistochemistry. We found that 5‐fluorouracil (5‐FU) induced autophagic cell death in HepG2 hepatocarcinoma cells by inhibiting PI3K/AKT/mTOR pathway. Orai1 expression was obviously elevated in hepatocarcinoma tissues. 5‐FU treatment decreased SOCE and Orai1 expressions, but had no effects on Stim1 and TRPC1 expressions. Knockdown of Orai1 or pharmacological inhibition of SOCE enhanced 5‐FU‐induced inhibition of PI3K/AKT/mTOR pathway and potentiated 5‐FU‐activated autophagic cell death. On the contrary, ectopic overexpression of Orai1 antagonizes 5‐FU‐induced autophagy and cell death. Our findings provide convincing evidence to show that Orai1 expression is increased in hepatocarcinoma tissues. 5‐FU can induce autophagic cell death in HepG2 hepatocarcinoma cells through inhibition of SOCE via decreasing Orai1 expression. These findings suggest that Orai1 expression is a predictor of 5‐FU sensitivity for hepatocarcinoma treatment and blockade of Orai1‐mediated Ca²⁺ entry may be a promising strategy to sensitize hepatocarcinoma cells to 5‐FU treatment.     

Linc01014 regulates gefitinib resistance in oesophagus cancer via EGFR‐PI3K‐AKT‐mTOR signalling pathway

This study aimed to explore the underlying mechanism of linc01014 in oesophagus cancer gefitinib resistance. Gefitinib‐resistant oesophagus squamous cell carcinoma (ESCC gefitinibR) cell lines were constructed by using different gefitinib treatment in FLO‐1, KYAE‐1, TE‐8 and TE‐5 cell lines and confirmed by MTS50 and proliferation assays. Expression of linc01014 was overexpressed/silenced in FLO‐1 cells followed by gefitinib treatment, and then, the apoptosis‐associated markers Bax and Bcl‐2, and PI3KCA in PI3K signalling pathway were determined using Western blotting. MST50 and morphology analyses showed that ESCC gefitinibR cell lines presented obvious gefitinib resistance than their parental ESCC cell lines. ESCC gefitinibR cell lines showed significantly higher proliferation abilities than their parental ESCC cell lines after treating with gefitinib. Overexpression of linc01014 significantly inhibited the apoptosis of FLO‐1 cells induced by gefitinib and silencing linc01014 obviously promoted the apoptosis of FLO‐1 cells induced by gefitinib. Silencing linc01014 could significantly increase the gefitinib chemotherapy sensitivity of oesophagus cancer via PI3K‐AKT‐mTOR signalling pathway.     

ω‐hydroxyundec‐9‐enoic acid induces apoptosis by ROS mediated JNK and p38 phosphorylation in breast cancer cell lines

ω‐Hydroxyundec‐9‐enoic acid (ω‐HUA), a plant secondary metabolite, exhibits anti‐fungal activity. However, its effect on breast cancer cells is unknown. Here, we investigated the anti‐ breast cancer activity of ω‐HUA and its underlying mechanism. Treatment of human breast cancer cell lines, MDA‐MB‐231 and MDA‐MB‐435, with ω‐HUA induced apoptotic cell death with increased cleaved caspase‐3 and poly (ADP‐ribose) polymerase (PARP) levels, and p38 and JNK phosphorylation. Inhibition of these mitogen‐activated protein kinase (MAPK) pathways using specific inhibitors or siRNA, for p38 and JNK, respectively, blocked the ω‐HUA‐induced apoptosis in a dose‐dependent manner. Moreover, pretreatment of the cells with antioxidant N‐acetyl cysteine (NAC) inhibited ω‐HUA‐induced increased reactive oxygen species (ROS) levels, cleaved caspase‐3 and cleaved PARP, and phosphorylated JNK, phosphorylated p38, and increased cell viability and colony‐forming ability. MDA‐MB‐231 xenograft model showed that the ω‐HUA‐treated group exhibited greater tumor regression and significantly reduced tumor weight compared to that exhibited by the vehicle‐administered group. Collectively, ω‐HUA‐induced intracellular ROS generation induced breast cancer cell apoptosis through JNK and p38 signaling pathway activation, resulting in tumor regression. The results suggested that ω‐HUA is an effective supplement for inhibiting human breast cancer growth.     

Inhibition of autophagy enhances apoptosis induced by the PI3K/AKT/mTor inhibitor NVP‐BEZ235 in renal cell carcinoma cells

The PI3K/AKT/mTOR pathway plays a key role in the development of the hypervascular tumor renal cell carcinoma (RCC). NVP‐BEZ235 (NVP), a novel dual PI3K/mTOR inhibitor, showed great antitumor benefit and provided a treatment strategy in RCC. In this study, we test the effect of NVP on survival rate, apoptosis and autophagy in the RCC cell line, 786‐0. We also explore the hypothesis that NVP, in combination with autophagy inhibitors, leads to apoptosis enhancement in 786‐0 cells. The results showed that the PI3K/AKT/mTOR pathway proteins p‐AKT and p‐P70S6K were highly expressed in RCC tissue. We also showed that NVP inhibited cell growth and induced apoptosis and autophagy in RCC cells. The combination treatment of NVP with autophagy inhibitors enhanced the effect of NVP on suppressing 786‐0 growth and induction of apoptosis. This study proposes a novel treatment paradigm where combining PI3K/AKT/mTOR pathway inhibitors and autophagy inhibitors lead to enhanced RCC cell apoptosis. Copyright © 2012 John Wiley & Sons, Ltd.     

PI3K inhibition potentiates Bcl‐2‐dependent apoptosis in renal carcinoma cells

Inhibitors of PI3‐K/Akt are currently being assessed clinically in patients with advanced RCC. Identification of therapeutic strategies that might enhance the efficacy of PI3‐K/Akt inhibitors is therefore of great interest. As PI3‐K inhibition would be expected to have many pro‐apoptotic effects, we hypothesized that there may be unique synergy between PI3‐K inhibitors and BH3‐mimetics. Towards this end, we assessed the combination of the PI3K inhibitor LY 294002 and the Bcl‐2 family inhibitor ABT‐737 in RCC cell lines. We found that the combinatorial treatment with these agents led to a significant increase in PARP cleavage and cell death in all RCC cell lines. The synergized cell death was correlated with decreased levels of Mcl‐1 and XIAP, and increased levels in Bim, and appears critically dependent upon the activation of caspase 3 and 8. The enhanced lethality observed with the combination also appears dependent upon the regulation of XIAP, Mcl‐1 and Bim levels. Our results suggest that the combination of PI3‐K inhibitors with BH3‐mimetics may be a viable therapeutic strategy in RCC.     

MiR‐29 mediates TGFβ1‐induced extracellular matrix synthesis through activation of PI3K‐AKT pathway in human lung fibroblasts

TGFβ1 is very important in the synthesis and degradation of extracellular matrix, and also in the mediation of human lung fibroblasts proliferation, and miR‐29 plays an important role in this process. To explore the interactions of miR‐29 family members and TGFβ1, the effects of transforming growth factor TGFβ1 on the expression of miR‐29 and whether miR‐29 is involved in pro‐survival signaling pathways mediated by TGFβ1 were examined in human lung fibroblasts. Treatment of the human embryonic lung fibroblast cell line IMR90 with TGFβ1 caused a decrease in expression of miR‐29a/b/c by real‐time PCR analysis. TGFβ1 stimulation increased cell proliferation, colony formation and up‐regulated expression of COL1A1; transfecting with miR‐29a/b/c mimics reverse TGFβ1‐induced phenotype changes in IMR90 cells. Western blot analyses showed that TGFβ1 treatment unchanged total protein expression levels of PI3K or AKT, but the expression levels of p‐PI3K, p‐AKT, and COL1A1 were increased; and miR‐19a/b/c mimics interfering blocked phosphorylation of PI3K or AKT and decreased expression of COL1A1 after TGFβ1 treatment. The results indicate that TGFβ1 beta uses the PI3k‐Akt pathway in these embryonic fibroblasts and miR29 blocks this activation pathway. It indicates a novel biological function of the PI3K‐Akt pathway in IMR90. Elevated expression of miR‐29 may play an important role in the pathogenesis of diseases related to fibrogenic reactions in human lung fibroblasts. J. Cell. Biochem. 114: 1336–1342, 2013. © 2013 Wiley Periodicals, Inc.     

Inhibition of PI3K/AKT signaling via ROS regulation is involved in Rhein-induced apoptosis and enhancement of oxaliplatin sensitivity in pancreatic cancer cells

Several natural products have been demonstrated to both enhance the anti-tumor efficacy and alleviate the side effects of conventional chemotherapy drugs. Rhein, a main constituent of the Chinese herb rhubarb, has been shown to induce apoptosis in various cancer types. However, the exact pharmacological mechanisms controlling the influence of Rhein on chemotherapy drug effects in pancreatic cancer (PC) remain largely undefined. In this study, we found that Rhein inhibited the growth and proliferation of PC cells through G1 phase cell cycle arrest. Moreover, Rhein induced caspase-dependent mitochondrial apoptosis of PC cells through inactivation of the PI3K/AKT pathway. Combination treatment of Rhein and oxaliplatin synergistically enhanced apoptosis of PC cells through increased generation of intracellular reactive oxygen species (ROS) and inactivation of the PI3K/AKT pathway. Pre-treatment with the ROS scavenger N-acetyl-L-cysteine attenuated the combined treatment-induced apoptosis and restored the level of phosphorylated AKT, indicating that ROS is an upstream regulator of the PI3K/AKT pathway. The combination therapy also exhibited stronger anti-tumor effects compared with single drug treatments in vivo. Taken together, these data demonstrate that Rhein can induce apoptosis and enhance the oxaliplatin sensitivity of PC cells, suggesting that Rhein may be an effective strategy to overcome drug resistance in the chemotherapeutic treatment of PC.